Summary of Work: Drosophila melanogaster maintains telomere length by a process of tip specific transposition. Two non-LTR retrotransposons, HeT-A and TART, are known to be responsible for telomere elongation in this organism. Although HeT-A is expressed in actively dividing cells, especially in the germ line, in wild type strains only about 1% of chromosome ends are extended per sexual generation, suggesting that transcription is not the limiting step in transposition. A few strains have terminal HeT-A arrays much longer than normal. A genetic factor on chromosome 3 has been identified that induces terminal HeT-A arrays to grow, but does not induce that transposition of non-telomeric retrotransposons. The long telomeres are found in telomere-telomere associations at a high frequency at mitosis. Unlike other retroposons, HeT-A doesn't encode its own reverse transcriptase (RT), a key enzyme in retrotransposition. The RT activity is higher in the strains with longer telomeres. To investigate the source of RT genetically and to show that this element transposes via an RNA intermediate as expected from its sequence, we have prepared constructs with HeT-A sequence and a reporter gene that will be expressed only after HeT-A transposition. Embryo injections to produce transformants are in progress. Once we can quantify transposition, these elements will be used to characterize the genetic factors that stimulate HeT-A transposition. A gene for Green Fluorescent Protein (GFP) will also be used as a reporter for transposition to relate cells expressing HeT-A mRNA to those that allow transposition. Similar constructs are being made using L1, a human non-LTR retrotransposon. A number of stably transformed cell lines are being used. Two of these cell lines are human colon cancer cell lines that are mutant for mismatch repair genes, hMSH6 and hMLH1. Another cell line mutant for p53 is under investigation. In p53 cells transposition occurs at a high frequency and there is a high incidence of apoptosis in cells with the active retrotransposon. This project is supported by an IRA.